PENINSULA LABORATORIES, LLCA MEMBER OF THE BACHEM GROUPGENERAL PROTOCOLFORPeptide Radioimmunoassay (RIA)CAUTION: INVESTIGATIONAL DEVICELIMITED BY FEDERAL LAW TO INVESTIGATIONAL USEFOR RESEARCH USE ONLYNOT FOR USE IN DIAGNOSTIC PROCEDURES305 OLD COUNTY RD. SAN CARLOS, CA 94070TEL: (650) 592-5392 (800) 922-1516 FAX: (650) 595-4071
2RIA KITS - BASIC NOTIONS AND FACTSOur RIA kits are competitive immunoassays whose working principle can bediagrammed as follows:A constant concentration of 125I-tracer (125I radiolabeled tracer) and varyingconcentrations of unlabeled standard or sample peptide compete for bindingspecifically to the antiserum. Immunocomplexes are precipitated by adding anexcess of non-specific serum and secondary polyclonal antiserum followed bycentrifugation. Unbound reagents are washed and the pellet is counted in agamma counter.The sequence of the standard peptide is shown on the datasheet. It is alsoin our catalog and on our web site (www.BACHEM.com) as the "antigensequence" (note that large protein sequences are usually not shown).The standard is used to make a standard curve in the range specified in thekits datasheet. Standard curves are S-shaped (on a semi-log plot) but fora few kits they appear to be almost linear over the kits range. The measuringrange is the range of standard concentrations near the middle or near the IC50of the standard curve. Typical standard curves for most kits are printed in ourcatalog but can also be obtained via e-mail (firstname.lastname@example.org).Unknown sample concentrations are measured by comparing its cpm with thestandard curve.We include sufficient reagents for 125 RIA test tubes.SpecificAntiserumStandard or Sample PeptideImmuno-precipitationCount pellet125I-Tracer
3VARIATION •••• ACCURACY •••• EXTRACTION •••• CROSSREACTIVITYThe kits IC50, or the shape of the standard curve, may show some variation butthis will not affect the kits accuracy in the measuring range. The kit accuratelymeasures sample peptides if the following conditions are met.A) The kits antiserum must not cross-react appreciably with otherfactors present in the sample. Cross-reactivity tables are included witheach kit. The user may wish to test the cross-reactivity with other peptides, someof which may be available from our catalog.B) The sample peptides must be identical to the kits standard.Ideally the kits synthetic standard mimics the natural peptide perfectly.Sometimes, however, natural peptides exist as families of species related by acommon or similar sequence. In addition, natural peptides may beenzymatically or spontaneously modified, may exist in complexes, and mayassume alternative structural forms. In these cases the kit might not measure theexact concentration of a particular natural peptide species, but, it may still beused for relative average measurements.C) Serum samples must be extracted. Factors present in serum can bindto kit components and affect the measurements. Thus, it is advisable to extractpeptides from serum before measuring them. Extraction may not be asimportant if the sample is diluted before measuring or if it is in tissue culturemedium, but, in general, one may expect low sensitivity or high backgroundproblems if extraction is not used. Some antigens can be measured directly inun-extracted serum. If so, a specially formulated extraction-free kit may beavailable in our catalog or as a custom product, which can be used withoutextraction for human or rat serum.D) Both samples and standards must be measured under the sameconditions.TECHNICAL HELP and ORDERING INFORMATIONPlease e-mail technical questions to email@example.comFor additional kits or listings of our most current catalog products, please visitour web site at www.bachem.com or call us at:Tel: (800) 922-1516 (for U.S. and Canada)(650) 592-5392 (for International)Fax: (650) 595-4071
4SAFETY PRECAUTIONSThe 125I radioisotope produces 35.5 KeV gamma radiation. It has a half-life of60.1 days, and a biological half-life of 120 to 138 days. The amount ofexposure from a 1.5 µCi tracer is 0.3 µR/Hr at contact. The critical organ foruptake is the thyroid gland. Please use shielding and equipment designed for125I or gamma radiation.This radioactive material shall be received, acquired, possessed and used bylicensed investigators in laboratories or hospitals for in vitro laboratory testsonly. Its use shall not involve internal or external administration of the materialand radiation therefrom to human beings or animals. Its receipt, acquisition,possession, use and transfer are subject to the regulations and licenserequirements of the U.S. Nuclear Regulatory Commission or of a State withwhich the Commission has entered into an agreement for the exercise ofregulatory authority.The user should designate an area for handling 125I and clearly label allcontainers. There should be no drinking, eating, or smoking where 125I is beinghandled. Use transfer pipettes, spill trays, and absorbent coverings to confinecontamination. All users should wear disposable lab coats, gloves, and wristguards as a secondary precaution. Regularly monitor and promptlydecontaminate gloves and surfaces to maintain contamination control. Use aNaI (TI) detector or liquid scintillation counter to detect 125I. Conduct periodicthyroid counts to determine an internal dose. Isolate waste in sealed, labeledcontainers.Establish surface contamination, air concentration, and thyroid burden actionlevels below maximum limits and investigate any causes that threaten theselevels to be exceeded. Persons under 18 years old should not be permitted tohandle radioactive material or enter radioactive areas.
5Disposal - Radioactive waste should be disposed of in compliance withFederal, State, and Local Government regulations.THIS PACKAGE CONFORMS TO THE CONDITIONS AND LIMITATIONSSPECIFIED IN TITLE 49 CFR173.421 FOR RADIOACTIVE MATERIAL.Some of the kit components (including the RIA buffer) contain Sodium Azide.Please handle and dispose of according to established safety regulations.GUARANTEE AND LIMITATION OF REMEDYPENINSULA LABORATORIES, LLC makes no guarantee of any kind, expressedor implied, which extends beyond the description of the material in this kit,except that these materials and this kit will meet our specifications at the time ofdelivery. The customer’s remedy and PENINSULA LABORATORIES, LLC’s soleliability hereunder are limited at PENINSULA LABORATORIES, LLC’s option torefund the purchase price or the replacement of material that does not meet ourspecifications. By the acceptance of our products, the customer indemnifies andholds PENINSULA LABORATORIES, LLC harmless against, and assumes allliability for the consequences of its use or misuse by the customer, its employeesor others.TRACER DECAY• Tracers should be used as soon as possible.• The specific activity of the 125I-tracer will decay with a half-life of 60.1 days(for example in 1 month the tracer will lose 30% of its radioactivity).
6MATERIALS PROVIDED AND STORAGE CONDITIONS• RIA buffer 4x concentrate 50 ml (Dilute to 200 ml). Store at 4°°°°Cbefore and after dilution.• Standard peptide lyophilized powder (for 1 ml RIA buffer). Store at 4°°°°Cbefore re-hydration and at -20°°°°C after rehydration.• Specific rabbit or guinea pig antiserum lyophilized powder (for 13ml RIA buffer). Store at 4°°°°C before re-hydration and at -20°°°°C afterrehydration.• 125I-tracer: at least 1.5 µCi (on preparation day). Store at -20°°°°C.• Secondary polyclonal antiserum lyophilized powder (for 13 ml RIAbuffer), goat anti rabbit (GARGG) or goat anti-guinea pig (GAGGG)antiserum depending on the kit. Store at 4°°°°C before and after re-hydration.• Normal serum lyophilized powder (for 13 ml RIA buffer). Normalrabbit or normal guinea pig serum depending on the kit. Store at 4°°°°Cbefore and after re-hydration.• Datasheet.• Standard Diluent 8 ml of peptide-free stripped frozen serum (forextraction-free kits only). Store at -20°°°°C.MATERIALS NOT PROVIDED• SEP-COLUMN containing 200 mg of C18 Cat No. Y-1000) (optional)• Extraction kit Cat No S-5000 (for 50 samples with buffers) (optional)• 12 x 75 mm polystyrene tubes• Gamma counter for the above tubes• Centrifuge for the above tubes• Curve fitting software (optional)• Various other standard laboratory items ...ASSAY PROTOCOLWe recommend using the same basic 3-day RIA protocol shown on thefollowing page for all our RIA kits. Depending on the kit, the details of theprotocol differ with respect to three parameters: extraction, standard curve,and antiserum type. Please refer to the enclosed datasheet to determinethese parameters and proceed as required for each step of the protocol.
8DAY 11 - Dilute the RIA buffer concentrate to 200 ml2 - Sample extraction is recommended especially for serum samples. It maynot be as important for some tissue culture samples. See "Suggested Protocolfor Sample Extraction" below for details. The kit may still be used withoutextraction but this may cause unexpected results due to the possible bindingbetween serum proteins and kit components.If you purchased an Extraction-Free Kit you may use it for themeasurement of human or rat serum or plasma (according to itsdesignation) without performing an extraction.Sample concentration. The concentration of the target molecule must bewithin the measuring range of the kit (in a region around the IC50). If youcannot estimate the concentration range of your sample you can prepare it atdifferent concentrations such that one of the samples may be within themeasuring range.3 - Reconstitute the standard in 1ml RIA buffer and make serialdilutions covering the kits range as stated in the encloseddatasheet.Caution: please note carefully the amount of standard provided and thekits range.The tables on the next page are included to facilitate the serial dilutionprocess.If you purchased an Extraction-Free Kit you should use the includedStandard Diluent - (8 ml of peptide-free stripped frozen human orrat serum) to make the dilutions.Note: the standards and the samples should be in the same diluent. Ifextraction was done the diluent should be RIA buffer. Otherwise, for extraction-free kits use your own diluent if possible or use the included standard diluent forstandards to match the serum or plasma samples.
94 - Reconstitute the antiserum in 13 ml RIA buffer.5- Mix 100 µµµµl standards or samples with 100 µµµµl antiserum.Add no standard or sample to controls TC, NSB, and TB.Add only 100 µl diluent and 100 µl antiserum to TB.Add 100 µl diluent plus 100 µl RIA buffer to TC and NSB.6 - Vortex and incubate overnight (16-24 hrs.) at 4°C.
10DAY 27- Prepare (a) the tracer stock and (b) the diluted tracer solutions.a) Tracer stock. Add 1 ml of RIA buffer to the shipped 125I-tracer vial andmix thoroughly.b) Diluted tracer. From the tracer stock make a working diluted tracersolution in RIA buffer at 10,000 to 15,000 cpm / 100 µµµµl. The kit will bemost sensitive with the least amount of tracer that will still provide accuratecounts at the lower end of the assay range. We have determinedempirically that 10,000 to 15,000 cpm per assay tube (TC cpm) workswell. If you need help in making the correct dilution of tracer please dothe following:o N = number of assays and standards (plus ~ 10%)o V = N x 0.1 ml (volume of needed RIA buffer in ml)o To = N x 2 µl (initial volume of tracer stock in µl)o Mix V ml RIA buffer with To µl tracer stock and count 100 µl• C100 = cpm in 100 µl this initial dilutiono Tadd = additional µl tracer that must be added to reach10,000 cpm/100µl100100 )000,10(CToCTadd −=If you require a higher specific activity than 10,000 cpm / 100 µl simplyenter a number greater than 10,000 in the equation above. If you requireless, enter a lower number and also start with a lower To. If C100 exceedsthe desired cpm dilute the initial solution in RIA buffer.The unused tracer stock solution can be stored at 4oC for no more than 1 day toavoid freezing and thawing. If storing for a longer period of time, createaliquots of the tracer to prevent multiple freezing and thawing and store at-20oC or lower for maximum stability.8- Add 100 µµµµl diluted 125I tracer (e.g. ~10,000 cpm) to all tubes.9- Vortex and incubate overnight (16-24 hrs.) at 4°C
11DAY 310, 11 - Add secondary Ab and normal serumMost of our RIA kits include goat-anti-rabbit-IgG (GARGG) as a secondary Aband normal rabbit serum (NRS) but a few include goat-anti-guinea-pig-IgG(GAGGG) secondary Ab and guinea pig serum (NGS).a) Reconstitute the secondary antibody (GARGG or GAGGG) with 13 ml ofRIA Buffer.b) Reconstitute the normal serum (NRS or NGS) with 13 ml of RIA Buffer.c) Add 100 µl of secondary antibody and 100 µl of normal serum to eachtube and vortex.12 - Incubate at room temperature for 90 minutes13 - Add 500 µl of RIA Buffer to each tube and vortex.14 - Centrifuge for 20 minutes at 1700 x g.15 - Set aside the TC tubes. DO NOT ASPIRATE THE TC TUBES.Aspirate all other tubes being careful not to disturb the pellet.16 - Count, plot and analyze the cpm data.Use a calibrated gamma counter and collect the cpm data for all tubes.Should you need help for the plotting and analysis of your data we recommendthe following procedure:Set up a spreadsheet as shown below (note that the values on the spreadsheetare merely illustrative and are not necessarily typical for this particular kit).Please e-mail us at firstname.lastname@example.org and we will be happy to sendyou the actual working Excel spreadsheet shown below.
12For tubes labeled TC, NSB, TB, H, G, F, ..., A, enter the cpm from the duplicatetubes and average the valuesCalculate all B/Bo values with the following formula:NSBTBNSBcpmsampleeAveragBoB−−=Plot a standard curve y = f(x) on a semi-log scale where y is B/Bo and x are thestandard concentrations in ng/ml.We recommend that you use a good automatic data fitting computer program,but you may also obtain excellent results by fitting a four-parameter curve to thestandard readings on a Excel sheet as shown here. Use the following equationto calculate the values on the "FIT" column. Then change the parameters a, b,c, and d, until you are satisfied that fit is good.dy += b(x/c)+1d-aNext calculate the average of your sample readings and from that calculateB/Bo for all your samples. Finally, you may use the "reverse" of the equationabove to calculate the concentrations in ng/ml for all your samples.bydaycx/1−−=
13Caution• In the example above we set the TB known ng/ml value at 0.001 so thatthe value could be plotted on a semi-log scale, but value is actually ZERO• When calculating sample concentrations using the "reverse" equation ifyour B/Bo value happens to be the same number as the parameter "d" youwill get a error because of a division by zero• If your B/Bo value is above or below the asymptotes obviously the readingis off range and the calculation wont workSUGGESTED PROTOCOL FOR SAMPLE EXTRACTIONWe have provided an excess amount of standard that you may use to determineif extraction is required. For example, if you are working with serum, you mayspike it with known amounts of standard and check if they are accuratelydetermined by the assay with and without extraction. Extraction eliminatespotentially interfering substances, such as albumin. Extraction may also benecessary to concentrate the sample to within the measuring range. As with anypurification technique, recovery of the desired substance is likely to beincomplete. Therefore, both optimization and quantification of the extraction
14procedure are recommended for more accurate determinations. While wecannot provide you with extraction optimization and quantification protocols, wehave included enough standard in the kit should you wish to use it for thispurpose.C18 Sep-Column Extraction MethodThe following generic protocol is meant to help users with little experience inextracting their samples. It should be applicable to different biological fluidsbut should not be thought of as an optimized protocol for any particularantigen.Required Materials• SEP-COLUMN containing 200 mg of C18 Cat No. Y-1000• Buffer A (BUFF-A): 1% trifluoroacetic acid (TFA, HPLC Grade). (Acidifiesplasma sample to remove interfering proteins such as albumin)• Buffer B (BUFF-B): 60% acetonitrile (HPLC Grade), 1% TFA, and 39%distilled water. (Elutes peptide from C18 column)• You may also consider purchasing Extraction kits (Cat No. S-5000), whichinclude SEP-columns and buffersWithdrawal and Preparation of Plasma• Collect blood samples (2 - 6 ml) into a chilled syringe and transfer into apolypropylene tube containing EDTA (1 mg/ml of blood) as ananticoagulant and Apronitin (500 KIU/ml of blood) as a protease inhibitorat 4°C. Do not use heparinized tubes as they may interfere with the assay.Vacutainers with EDTA are acceptable.• Centrifuge blood at 1,600xg for 15 minutes at 4°C.• Collect the top (plasma) layer.• Proceed to extraction immediately or freeze at -70°C for later use.Extraction Procedure• Add an equal amount of Buffer A to the plasma.• Centrifuge at 6,000xg to 17,000xg for 20 minutes at 4°C.• Transfer supernatant to a new tube discarding any pellet that may be
15present.• Equilibrate a SEP-COLUMN by washing with 1 ml Buffer B followed by 3 X3 ml Buffer A.• Load the plasma solution onto the equilibrated SEP-Column.• Slowly wash the column with Buffer A (3 ml, twice) and discard the wash. Alight vacuum (10 sec/drop) may be applied to the column.• Elute the peptide slowly with Buffer B (3 ml, once) and collect eluant in apolypropylene tube. A light vacuum may be applied as in previous step.• Freeze-dry eluant to dryness using a dry ice/methanol bath to freeze thesample and a centrifugal concentrator to evaporate it• Dissolve the residue in a suitable volume of RIA buffer such that theconcentration of the substance of interest will fall close to the IC50 (withinthe measuring range).TROUBLESHOOTING.Most problems are caused by poor technique or by alterations of the protocol.What follows are possibly less obvious causes of problems.Poor precision (high standard deviations) may be caused by• Poor mixing• Kit reagents not allowed to equilibrate to room temperature before use.• Cross-contamination of samples• Insufficient centrifugation time or g forcePossible causes of low bindingBo or "total tpecific tinding" is obtained by subtracting the non-specificbinding cpm (NSB) from the max binding cpm (TB): Bo = TB - NSB."Total binding" is the percent Bo relative to "total cpm" (TC)Total Binding = 100 x Bo / TC"Low binding" occurs if "total binding" is below 25%. This leads to shallowcurves and poor precision.Low binding can be caused by:• Degraded tracer or antiserum due to incorrect storage or excessivefreeze-thawing• Reduced incubation time
16Possible causes of nonspecific binding• Tracer may be sticking more than expected to a particular test-tube. Makesure you add 500 µl RIA buffer before vortexing and centrifuging. Also,test different types of tubes.• High incubation temperature may cause higher sticking.• Contamination with antiserumPossible causes for higher than expected IC50• Note that the IC50 reported with each of our products is based on theconcentration of the prepared standards before they are added to theassay solution.• A difference by a factor of two or three may be normal.• In cases where the standard curves are almost rectilinear, accurate IC50values cannot be calculated with the 4-parameter equation shown above.In these cases it is best to estimate the IC50 visually as the x value thatresults in a B/Bo of 0.50.• Excessive amounts of antiserum or tracer, or using a degraded standardmay all raise the IC50.Poor standard curves may be caused by• Loss of precipitate - use appropriate g force and time for centrifuge.• Aspirate immediately after centrifuging.• Do not leave excessive liquid behind.• Many other trivial explanations: bad pipettes, etc.Unexpected results may be obtained if• Standards and samples are in different solvents or conditions• Antiserum binds to another antigenically similar peptide• Antigen was lost during extraction or extraction did not eliminateinterfering factors (not applicable to extraction-free kits)REFERENCES1. Patrono, C. and Peskar, B.A. (eds) Radioimmunoassay in Basic andClinical Pharmacology. Heidelberg, Springer-Verlag, 1987.2. Dwenger, A. Radioimmunoassay: An Overview. J Clin Biochem 22:883,1984.